The research outlined in this proposal is directed towards the general question of how membrane proteins are properly sorted and localized to specific eukaryotic cellular organelles. The biogenesis and maintenance of organelles, a poorly understood process dependent on correct localization and retention of membrane proteins, is critical for proper cell function. In addition, several human diseases (e.g. cystic fibrosis and familial hypercholesterolaemia) arise due to defects in the sorting of membrane proteins. While many membrane protein sorting events have been identified in eukaryotic cells, little is known of the machinery responsible for recognizing and localizing membrane proteins to the correct cellular compartment. This proposal describes a multi-faceted approach to characterize the mechanism by which the Golgi apparatus retains its resident Golgi membrane proteins. This analysis has been conducted in the yeast, S. cerevisiae, because this eukaryotic organism is easily amenable to genetics. The late Golgi enzyme, dipeptidyl aminopeptidase (DPAP) A, has been shown to contain a retention signal it its cytoplasmic domain which is both necessary and sufficient for its localization in the Golgi apparatus. In order to identify the cellular machinery that recognizes this signal and retains DPAP A in the Golgi, a genetic screen has been developed to identify mutants which fail to properly retain their Golgi membrane proteins. The grd (Golgi retention defective) mutants identified thus far fall into 10 complementation groups. The elucidation of the role of the individual grd gene products in the retention of Golgi membrane proteins promises to illuminate the Golgi retention mechanism and in particular may provide the basis for identification of the DPAP A retention signal receptor. Independent genetic and biochemical approaches will also be pursued that more directly focus on identification of the retention signal receptor. Experiments to determine whether or not retention occurs via retrieval from a post-Golgi compartment will also be performed. The results of these studies should help illuminate the mechanism of yeast Golgi membrane protein retention and add significantly our overall knowledge of membrane protein sorting.